Summary. Incorporation of 15NO3- into amino acids was studied during the anaerobic germination of rice seeds. In treated coleoptiles, the label was.
Amino Acids (1995) 9:385-390
Amino Acids
© Springer-Verlag1995 Printed in Austria
Incorporation of nitrate nitrogen into amino acids during the anaerobic germination of rice
Short Communication R. Reggiani 1, F. BertinF, and M. Mattana ~ l Istituto Biosintesi Vegetali and 2Istituto Chimica Macromolecole, C.N.R., Milano, Italy Accepted June 6, 1995
Summary. Incorporation of 15NO3- into amino acids was studied during the anaerobic germination of rice seeds. In treated coleoptiles, the label was incorporated into glutamine, glutamate, alanine, 7-aminobutyric acid (Gaba), arginine, aspartate and methionine. These findings are consistent with a primary incorporation of nitrate nitrogen into glutamine, glutamate and aspartate, and their further conversion to alanine, Gaba, arginine and methionine.
Keywords: Amino acids - Anoxia - Coleoptile - Nitrate nitrogen - Rice Introduction Rice seeds germinating in flooded soils may be subjected to anaerobiosis. Unlike most higher plants, rice is able to germinate in a strictly anoxic environment emitting only a white coleoptile (Opik, 1973). The anaerobic elongation of rice coleoptile is sustained by a continuous translocation of carbohydrates, amino acids and salts from the endosperm (Atweel et al., 1982). This flux of nutrients furnishes fermentable substrates and maintains turgor pressure at high levels for cell enlargement. Amino acids, among which alanine and 7-aminobutyric acid (Gaba) are the most representative (Reggiani et al., 1988), are important in maintaining the osmotic pressure in the anaerobically grown coleoptile (Menegus et al., 1984). Recently, it has been established that nitrate ions are translocated from the caryopses (Reggiani et al., 1993b,c) and assimilated by the rice coleoptile during the anaerobic germination (Mattana et al., 1993). The enzymes of nitrate reduction (nitrate and nitrite reductases) and ammonia assimilation (cytosolic and plastidial glutamine synthetase) are anaerobically expressed in the coleoptile (Mattana et al., 1994a,b). In the rice coleoptile, most of the
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nitrogen (both nitrate and a m m o n i u m ) was channelled toward the synthesis of alanine and G a b a u n d e r anaerobic conditions, while a r e d u c e d a m o u n t of nitrate was incorporated into these two amino acids in air (Fan, 1994). The biosynthesis of m a n y protein amino acids is compartimentalized into the plastids (Singh and Matthews, 1994) and, in the rice coleoptile, the amino acid d e m a n d for protein synthesis is as high in anoxia as in aerobic conditions (Aurisano et al., 1995). The objective of our work was to investigate the incorporation of nitrate nitrogen into the different amino acids during the anaerobic germination of rice.
Materials and methods Seeds of rice (Oryza sativa L. var. Arborio) were sterilized and anaerobically germinated for 6 days following the method previously adopted (Reggiani et al., 1993b). Then, 1mMKISNO3 was added for 2 additional days and the coleoptiles collected for analysis. Perchloric acid extracts and amino fractions were obtained as previously described (Reggiani et al., 1993b). The amino acid composition of the extract was determined by HPLC analysis of the o-phtaldialdehyde (OPA) derivatives according to Reggiani et al. (1993a). The separation of OPA derivatives was performed at a flow rate of 1.0ml/min on a 250 × 4mm Daltosil 100 ODS2 (4/~) reverse-phase column (Serva). Two mobile phase were used: A) 50mM Na-acetate buffer (pH 6.5) -tetrahydrofuran (97:3 v/v); B) methanol. Phase B was linearly increased from 0% to 70% in 35min. The amino acid fraction was freeze-dried before derivatization for gas chromatography-mass spectrometry (GC-MS) analysis. Appropriate amounts of the freeze-dried amino acid fraction (ca. 1.5mg) were placed into l ml screw-cap derivatization vials. Thirty /A of trifluoroacetic acid (TFA) and 150/A of N-(tert.-butyldimethylsilyl)-Nmethylfluoroacetamide (MTBSTFA) were sequentially added to the vials at 0°C. The reaction mixture was then maintained at room temperature for lh. Aliquots (1-2/A) of the solution containing the derivatives were injected directly into the gas-chromatograph. In order to improve the conditions for the derivatization of the more basic amino acids, histidine, lysine and arginine, N,N-dimethylformamide (DMF) was used as solvent in place of TFA. In these experiments the vials were heated at 150°C for 2 h. Derivatized samples were injected into a Hewlett-Packard 5985 B GC-MS apparatus operating in electron impact (EI) mode. The gas-chromatograph was equipped with a SUPELCO SPB-50 fused silica capillary column (30m × 0.32mm i.d., 0.25/~m film thickness). The oven was programmed from 130 to 270°C at 7°C/rain holding the initial temperature for 5 min. Mass spectral data were obtained under the following conditions: ionizing electron energy, 70eV; emission current, 0.3mA; ion source temperature, 200°C; scanning rate 1.5scan/s over the mass range m/z 50-600. The tert.-butyldimethylsilyl-amino acids (tBDMS-AA) derivatives possess characteristic mass spectra with intense diagnostic ions, generally at (M-57) +. This makes them very useful for selected ion monitoring (SIM) GCMS experiments. 15Nincorporation was calculated after integrating the areas obtained for selected ion for both labeled and unlabeled amino acids and expressing the data as atom % excess (Robinson et al., 1991). ~SN incorporation was considered not significant for amino acids exhibiting atom % excess 1 was considered significant; 2not significant
might be higher into G a b a t h a n into alanine. This fact could be associated with the increase in t h e level of G a b a o b s e r v e d in rice seedlings s u p p l e m e n t e d with 1mMK15NO3 (Table 1). B o t h alanine and G a b a are synthesized in the cytoplasm suggesting that m o s t g l u t a m a t e left the plastid after a m m o n i a assimilation ( t h e r e f o r e a - k e t o g l u t a r a t e has to be i m p o r t e d into the plastid). H i g h 15N i n c o r p o r a t i o n was also o b s e r v e d into arginine (394 pmol/coleoptile), suggesting that the m a r k e d effect of nitrate ions on arginine level (Table 1) m a y be attributed to a stimulation of its biosynthesis f r o m glutamate. A r g i n i n e is an i m p o r t a n t a m i n o acid in the a n a e r o b i c rice coleoptile, being p r e c u r s o r for the synthesis of putrescine, a c o m p o u n d involved in the elongation of the coleoptile (Reggiani et al., 1989). T h e p a t h w a y of arginine biosynthesis f r o m g l u t a m a t e is restricted to plastids, with the conversion of citrulline to arginine occurring in the cytoplasm (Bryan, 1990). Label was also d e t e c t e d into aspartate a n d m e t h i o n i n e (the latter belonging to the aspartate family). T h e 15N i n c o r p o r a t i o n into m e t h i o n i n e ( l l 2 p m o l / c o l e o p t i l e ) , alt h o u g h 4 - 5 times lower t h a n the i n c o r p o r a t i o n into alanine and Gaba, would indicate a significant a n a e r o b i c synthesis of this a m i n o acid. This last d a t u m is s t r e n g t h e n e d by the label into aspartate, whose precursor. Since m e t h i o n i n e is synthesized in the c y t o p l a s m f r o m h o m o c y s t e i n e p r o d u c e d in the plastid
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(Anderson, 1990), aspartate is likely synthesized from glutamate by the plastidic aspartate aminotransaminase. The role of methionine in the anaerobic metabolism of the rice coleoptile is still uncertain. These data taken as a whole would indicate that glutamine, glutamate and aspartate are the primary products during the assimilation of the nitrate nitrogen. The final fate for the nitrate nitrogen in the anaerobic rice coleoptile would be the incorporation into alanine, Gaba, arginine and methionine.
Acknowledgements Research supported by National Research Council of Italy. Special Project RAISA, subproject No. 2, paper No. 2143.
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Reggiani R, Mattana M, Aurisano N, Bertani A (1993c) The rice coleoptile: an example of anaerobic nitrate assimilation. Physiol Plant 89:640-643 Robinson SA, Slade AP, Fox GG, Phillips R, Ratcliffe RG, Stewart GR (1991) The role of glutamate dehydrogenase in plant nitrogen metabolism. Plant Physiol 95:509-516 Singh BK, Matthews BF (1994) Molecular regulation of amino acid biosynthesis in plants. Amino Acids 7:165-174 Authors' address: Dr. R. Reggiani, Istituto Biosintesi Vegetali, C.N.R., via Bassini 15,
1-20133 Milano, Italy. Received April 7, 1995
